The unifying goal of this proposed research is to elucidate certain mechanisms of CO2 kinetics in red cells. We will test the hypothesis that the direct transfer of protons from the carbonic anhydrase active site to buffers in solution is of physiological importance in systems which require a rapid supply of protons: 1) the transfer of protons to hemoglobin to produce the Bohr effect and 2) the enhancement of CO2 transport across thin aqueous layers by carbonic anhydrase and buffers. This will be carried out by observing the effect of buffers including hemoglobin on the carbonic anhydrase-catalyzed exchange of 18O between CO2 and water. 18O exchange will also be used to measure the inhibition of carbonic anhydrase by anions. This inhibition as measured by initial velocity techniques has produced data which are controversial and difficult to interpret. This topic will be clarified by our equilibrium experiments to determine inhibition constants and mode of inhibition by anions in the presence of the equilibrium ratio of CO2 and bicarbonate. We will extend these studies to include the inhibition of red cell carbonic anhydrase by intracellular anions, to assess the activity of intracellular carbonic anhydrase, and possibly to elucidate the function of human carbonic anhydrase B. 13C NMR relaxation measurements will be used in obtaining a value for the permeability constant of the red cell membrane to CO2. A major objective is to compare this value with that obtained by our 18O-exchange method, which gives a value for this constant which appears to be low compared to results of other experiments. Again relying on 18O exchange techniques developed in this lab, which give precise and reasonable values for the permeability constant of Bicarbonate across the red cell membrane, we will quantitate the effect on bicarbonate flux into red cells of anion diffusion inhibitors. Included will be those inhibitors found effective in blocking chloride and bicarbonate; also included will be certain sulfonamides which have been found to inhibit the flux into red cells of chloride and simple organic anions.